The Se-containing hydrogenase was purified to near homogeneity from extracts of Methanococcus vannielii. The total contents of trace elements in the enzyme were determined by atomic absorption spectroscopy. These are 3.8 g atoms of Se, 2 g atoms of Ni and 18-20 g atoms of Fe per mole of enzyme (Mr = 340,000). The subunit composition of the enzyme was estimated as Alpha2Beta4Gamma2 (Alpha = 56,000, Beta = 42,000 and Gamma = 35,000 Mr). Amino acid composition of the hydrogenase was also determined. The purified enzyme catalyzes reduction of the natural 8-hydroxy-5-deazaflavin cofactor with molecular hydrogen. When this reaction was carried out in tritiated water, negligible tritium label was detected in the dihyro-5-deazaflavin fraction. Kinetic values of this reaction were determined as Km = 16.7 MuM, kcat = 126,000/sec. Stereochemical studies demonstrated that the Se-containing hydrogenase and 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from M. vannielii recognize the same side with respect to the prochiral center at C-5 of dihydro-8-hydroxy-5-deazaflavin cofactor. To study the absolute configuration at C-5 of the reduced cofactor, analogues of 5-deazaflavin cofactor were reduced by the hydrogenase under molecular hydrogen in a large scale experiment. The selenium-independent formate dehydrogenase (Mr\= 100,000) was purified to near homogeneity from extracts of M. vannielii. The subunit composition of the enzyme was determined as AlphaBeta (Alpha = 60,000, Beta = 33,000 Mr.).